Aryl thioglycoside-based affinity purification of exo-acting cellulases.
The influence of ligand-coupling chemistry and mobile-phase composition on the interaction of exo-acting cellulases with an immobilized complementary ligand was investigated. p-Aminophenyl 1-thio-beta-D-cellobioside (APTC) was used as a representative affinity ligand to which exo-acting cellulases (cellobiohydrolases, CBHs) preferentially bind. A "crude" cellulase preparation from the fungus Trichoderma reesei served as an enzyme source. The adsorption properties of the two principal exo-acting CBHs in this preparation, CBH I and CBH II, are shown to be distinctly different under several scenarios. Their relative affinities, based on column elution behavior and partition equilibrium experiments, are shown to be highly dependent on the functional groups employed for ligand coupling, the extent of functional group hydrolysis, the composition of the mobile phase, and the inherent nature of the enzymes. The dependency on the chemistry of the supporting matrix was illustrated using agarose supports containing cyanate ester, N-hydroxy-succinimide, and epoxy functional groups. When compared under apparent optimal conditions, the affinity of CBH II for immobilized APTC was approximately 10-fold that of CBH I. However, selective adsorption of CBH I or CBH II can be achieved by adjusting experimental parameters.
Piyachomkwan
K
K
Department of Food Science and Technology, Oregon State University, Corvallis 97331-6602, USA.
Penner
M H
MH
D001487:Basidiomycota / Q000201:enzymology; D002475:Cellobiose / Q000031:analogs & derivatives*; D002480:Cellulase / Q000302:isolation & purification* / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity / Q000379:methods; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D012685:Sepharose
C111045:4-aminophenyl-1-thiocellobioside; D005656:Fungal Proteins; D008024:Ligands; D002475:Cellobiose; D012685:Sepharose; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase